Project Summary Elucidating the Mechanism of B-Raf Dimerization Inhibition Using (+)-Griffipavixanthone Derivatives Ras, Raf, MEK, and ERK are common hotspots for oncogenic expression as they are proteins involved in a highly-regulated pathway controlling cell proliferation. A single mutation can result in a hijacked cell exponentially replicating without regulation. Developing tools to probe, understand, and prevent the effects of these mutations are of great interest as there are currently no FDA-approved drugs that inhibit mutant Ras, an oncogene accounting for approximately 1/3 of all human cancers. The FDA has approved few inhibitors of mutant B-Raf; however, life expectancy is extended for only 3-8 months as resistance is acquired. (+)-Griffipavixanthone (GPX), a dimeric xanthone natural product that we can readily access by asymmetric synthesis, has demonstrated anticancer activity in various cell lines. In particular, we have found maximal potency in cancer cells with K- RasG12 and B-RafV600E mutations. When compared to the FDA-approved drug, sorafenib, GPX demonstrated a lower GI50 and similar or higher LC50. We have found that B-Raf dimerization, an important event mediated by Ras in cell proliferation, is inhibited upon treatment with (+)-GPX. Interestingly, the response is greater in (+)-GPX than its unnatural (-) enantiomer with a long response time for inhibition of B-Raf dimerization (18 h). Objectives: The proposed study will (1) expand on the key asymmetric reaction and employ late-stage functionalization to generate a compound library to understand the delayed response time of B-Raf dimerization inhibition, as we believe that GPX is likely a prodrug that undergoes intracellular oxidation to a reactive p- quinone methide. These advancements would have considerable impact on drug development and inhibitor design, and in the chemistry community; (2) ascertain the mechanism of action as it attenuates a deregulated, hijacked pathway. This will have high broader impact on the community and those studying this oncogenic pathway; (3) elaborate GPX to be more potent, drug-like, and target specific. Three aims are proposed to address the aforementioned objectives. Aim 1: A library of analogues with varying functionality, GPX-derived prodrugs with increased lipophilicity, and oxidized variants will be synthesized and evaluated in a time-dependent manner for B-Raf dimerization inhibition. Aim 2: A complete characterization of intracellular effects and downstream signaling by GPX and its derivatives will be performed in WT and Raf/Ras mutant cells. We will also study direct GPX-Ras/Raf surface binding in vitro and in vivo. Additionally, an unbiased pull-down experiment will be used to confirm intracellular targets. Aim 3: We will develop structural models of GPX derivatives and candidate receptors, including K-Ras mutants, and use this information to design higher affinity analogues. Overall, this research will impact chemical synthesis, prodrug development and metabolomics, and will provide important information on and tools for study of Ras/Raf/MEK/ERK, the most deregulated oncogenic pathway.